Zinc finger nucleases (ZFN)-mediated CCR5 disruption has shown to be a promising strategy for HIV gene therapy, but the latent viral reservoir still remains the major obstacle towards sterilizing or even a functional cure for HIV-1 infection. Resting CD4+ memory T cells are thought to be the major source of latency, and these also constitute a particularly stable reservoir of virus. ZFN targeting the conserved sequences in the 3' and the 5' LTR was reported to precisely excise the entire provirus in J-Lat cells, but the difficulty in delivering ZFNs to resting primary CD4+ T cells limited the potential f this approach for latency reduction. Recently, we have developed a method by using HIV env pseudotyping non-integrating lentivirus, successfully delivered CCR5-ZFN to resting CD4+ T cells and showed effectiveness in vivo in Hu-PBL mice. We have also found in preliminary studies that using Sindbis virus glycoprotein engineered to express ZZ-domain (Fc binding domain from protein A) provides another easy way to target resting CD4+ T cells. Here the pseudotyped lentivirus is treated with CD4 antibody (the ZZ domain containing lentivirus can bind any antibody) before infecting T cells. We could demonstrate targeting in >70% of resting CD4+ T cells. Thus we propose to target resting CD4+ T cells with non-integrating lentivirus carrying LTR and/or CCR5 ZFNs via HIV env-pseudotyped or Sindbis virus env-ZZ-domain, by which it would enable easy gene editing. We will use this method to edit CCR5 and/or HIV-LTR. Therefore, CCR5 disruption in bystander CD4+ T cells would protect uninfected cells and LTR/ZFN would excise integrated provirus in infected primary CD4+ T cells. The combination of these two strategies would potentially provide complete HIV protection. The long-term goal of this proposed research is to develop an alternative effective and safe HIV therapy that might offer a functional cure with simplified regimen. We propose to: 1) Generate and optimize reagents for Zinc Finger Nuclease (ZFN)- based gene editing in resting CD4+ T cells; 2) Evaluate whether ex vivo ZFN-modified PBMCs from ART suppressed individuals are protected from reactivation of endogenous virus after reconstitution in Hu/PBL mice